Specific rolling circle amplification of low-copy human polyomaviruses BKV, HPyV6, HPyV7, TSPyV, and STLPyV

•Development of a RCA method to specifically amplify low-copy polyomavirus genomes.•BKV genome could be amplified 1×108-fold from as little as 10 copies in mixed DNA.•Clinical TSPyV, HPyV6, HPyV7, and STLPyV positives were enriched up to 1×1010-fold. Eleven new human polyomaviruses have been recently discovered, yet for most of these viruses, little is known of their biology and clinical impact. Rolling circle amplification (RCA) is an ideal method for the amplification of the circular polyomavirus genome due to its high fidelity amplification of circular DNA. In this study, a modified RCA method was developed to selectively amplify a range of polyomavirus genomes. Initial evaluation showed a multiplexed temperature-graded reaction profile gave the best yield and sensitivity in amplifying BK polyomavirus in a background of human DNA, with up to 1×108-fold increases in viral genomes from as little as 10 genome copies per reaction. Furthermore, the method proved to be more sensitive and provided a 200-fold greater yield than that of random hexamers based standard RCA. Application of the method to other novel human polyomaviruses showed successful amplification of TSPyV, HPyV6, HPyV7, and STLPyV from low-viral load positive clinical samples, with viral genome enrichment ranging from 1×108 up to 1×1010. This directed RCA method can be applied to selectively amplify other low-copy polyomaviral genomes from a background of competing non-specific DNA, and is a useful tool in further research into the rapidly expanding Polyomaviridae family.